Previously we identified and characterized a family of interspersed 400 bp long repetitive sequences (R-sequences) representing 1-2% of mouse genomic DNA. We have now studied the functional role of these sequences in RNA transcription. Plasmid constructs containing R-sequence and the bacterial gene chloramphenicol acetyl transferase have been used in transient expression assays to measure promoter and enhance functions after transfection into mammalian cells. Several R-sequences increased transcription from the SV40 early promoter in monkey cells, and one R-sequence also increased transcription from an intracisternal A-particle long terminal repeat promoter when present 5 feet to the promoter. Polyadenylated RNA transcripts containing R-sequence have been detected in normal (thymus) as well as transformed (neuroblastoma) cells.